The present invention relates generally to an automated apparatus and method for performing assay testing on specimens, such as biological specimens. More specifically, the invention is directed to an automated apparatus and method for assay procedures to detect the compatibility of tissue or blood from a donor to a recipient.
Modern test procedures for determining or measuring the optical or electrochemical development of unknown specimens are used extensively in a number of medical testing procedures. In such tests, sample specimens are reacted with reagents and other substances. Such known procedures involve a variety of different assay steps but typically rely on detection and measurement of optical changes in a sample or label during the assay procedure. For example, a number of well known procedures use single or multi-wavelength fluorescence. These and other immunoassay techniques are known as Fluorescence Polarization Immunoassay (FPIA), solid phase agglutination, stained cellular morphology, Enzyme Immunoassay (EIA), chemolumination, spectrophotometric assays.
Other currently used assay techniques are effected by exposing the resulting sample to either transillumination or reflectant illumination. These assay procedures involve detecting the intensity of colorization, detecting ratio of multiple wavelengths of colorization, detecting the polarization in the sample, determining the size and quantity of specific cells at certain wavelengths, the general cell morphology or other optical characteristics of the results. The data from these procedures is then processed in a known manner to obtain the concentration or ratio of the component (or components) of interest. These techniques, however, have not been completely accepted and usually manual analysis is also performed as a check or verification.
One assay procedure of particular interest is a procedure known as Human Leukocyte Antigen (HLA) typing. This procedure is employed in matching tissue, body organs or blood from a donor to a recipient. In this HLA procedure, lymphocytes in samples containing human cells are first reacted with different antisera. The cell-serum mixture is then incubated with a complement. One or more stains are added to the mixture, with one of the stains staining dead cells. The reactions are then evaluated by calculating the ratio of dead cells (lyced cells) to live cells. The calculations are performed by using a microscope and estimating the ratio. This ratio is converted into a "score" ranging from 1-8 by using a well-known value scale.
In this HLA procedure, as well as other assay procedures, paramagnetic particles are coated with an antibody. The paramagnetic particles are then mixed with a sample to be analyzed. The antibody on the paramagnetic particles binds to specific cells in the sample. These specific cellular components may then be separated from the other cells in the population which is being tested using magnetic separation techniques. Alternatively the cells may be separated by using a nylon wool column. After the cells have been reacted with the antibody, the sample mixture is subjected to a series of operations such as particle exposure, reagent exposure, incubation, and washing. The cells in the sample may also be stained with one or more chemical markers as discussed above with respect to HLA assays. The sample is then analyzed. Typically, the sample will be analyzed manually by the technician. This manual analysis usually involves a visual analysis to determine the approximate percentage of the cells which have reacted with the antibody.
A significant shortcoming of these and other available assay techniques is that most of the steps in the procedure must be performed manually. For example, most of these procedures require manual preparation of the sample. Further, steps such as dispensing, mixing, washing, incubation, data collection, scoring and recording are also performed manually. Thus, most available assay techniques require a significant amount of human operator time.
As will be apparent to those skilled in the art, manual performance of these steps is also undesirable since it results in numerous opportunities for errors to occur. This is especially true for highly repetitive functions. The probability of errors is further amplified by the fact that many of these procedures require pipetting of very small volumes, i.e. usually of sub-microliter volumes. Further, scoring of thousands of reactions using a microscope and pencil also increase the probability of errors in the analysis.
A further drawback is the subjectivity which is permitted to the individual performing the test. This subjectivity may lead to inconsistent results, not only from assay to assay, but inconsistent analysis during the numerous repetitions in the same assay.
Although some available HLA assay devices automate individual steps, most of the steps in these devices are still performed manually. For example, U.S. Pat. No. 4,318,886 (Kawahara et al.) discloses an apparatus for HLA typing which uses a phase-contrast microscope and an optical image to generate a signal which is detected by an electrical signal pickup unit. The image is then binarized and compared with predetermined template patterns corresponding to reacted or non-reacted lymphocyte. Although the scoring of the results is automated, the preparation, incubation and washing of the sample must still be performed manually by the operator.
Further, this apparatus uses dedicated electronic hardware to score the HLA typing test. As discussed in more detail below, anomalies such as dirt or dust in the sample, scratches in the sample container, or unusually large cells would result in unreliable or erroneous results. In fact, without redesign for such possible variations, many human readable samples are unreadable by this apparatus. Variations in the procedures used by the operator preparing the samples may also lead to unreliable results without major redesign of the system. In summary, any expansion of the apparatus to score assays other than those it was specifically designed for is difficult and costly, requiring major redesign of the hardware for each assay.
Another major disadvantage of available automated systems, such as the one disclosed in U.S. Pat. No. 4,318,886, in that they are designed for a specific assay procedure (such as HLA typing). It is not possible to perform assays for which the instrument was not originally designed without major redesign of the hardware and or software. Such major redesign is impracticable and thus the use of available instruments is limited to a single type of assay.
Precise dispensing of the sample in reaction wells is also critical for accurate assay results. In HLA typing the dispensing is usually performed manually. A typical manual dispensing operation may include dispensing sample volume of from 0.5 .mu.l to 1.0 .mu.l into a volume of from 0.5 .mu.l to 2.0 .mu.l of a reagent which is covered by from 2.5 .mu.l to .mu.l of mineral oil. (The oil is used to prevent evaporation of the reagents.) It will be appreciated that performing this dispensing step involves a significant amount of operator time, which increases as the number of different reagents increases. Further, the operator will usually insert the tip of the pipette below the bottom surface of the oil and into the reagent itself. In order to prevent carryover from one reaction site to the next, the operator will typically manually wipe the tip of the probe thus consuming more operator time and increasing the chances for erroneous results.
If automated assay apparatus and methods are to be used in HLA assay procedures, they must be capable of very precise monitoring of liquid levels and precise control of liquid dispensing mechanisms (such as a pipette). Precise dispensing mechanisms are particularly important in HLA typing since, as discussed above, very small volumes of liquids (sub-microliters) have to be dispensed usually into a container which contains another liquid. Although some automated liquid dispensing systems are presently available, they are not completely suited for dispensing liquids in assays such as HLA typing. Available automated liquid dispensing systems usually work by detecting the liquid level in a container and then determining the position of the dispensing probe relative to the liquid surface. This information is then used to determine when the probe tip is within the liquid in the sample container. After the liquid surface has been detected and it has been determined that the probe is in the fluid, fluid may be dispensed into or aspirated from the container. The precision of the liquid dispensing system will thus depend in part on the precision of the liquid level detection.
The limited potential for available liquid level detection and fluid dispensing systems in HLA assay typing is due to the fact that they typically use a capacitance method to detect the liquid surface as a pipetting probe moves towards the liquid in a sample container. Dispensing liquids in volumes smaller than one microliter is complicated in such capacitive or conductance systems since the oil which covers the reagent has a low dielectric constant. The dielectric constant of oil is only two times greater than the dielectric constant of air rendering most capacitive detection methods unreliable for detection of the oil surface. Further, because of the high resistivity of the oil, available conductance methods cannot be used accurately.
Available capacitive type dispensing systems also do not have any means for determining when a droplet of the sample is formed on the dispensing probe or when a droplet of the sample has been separated or released from the probe tip. The ability to detect the occurrence of one or both of these events is important information which could be used to improve the accuracy and reliability of the dispensing system.
Therefore, it would be desirable to have a liquid level detection and liquid dispensing arrangement capable of detecting very small amounts of liquid (down to fractions of a microliter) and with the capability of detecting the level of a liquid having a low dielectric constant.
Another area of assay testing where significant improvements are necessary is in the area of image processing used for counting reactions. Although photomultiplier tubes have been previously used in some HLA readers, they are not without disadvantages and have not been readily accepted in the market. These readers use the photomultiplier tube as a fluorescent densitometer to measure the overall light output from the reaction site for each wavelength. This is acceptable for ideal samples but produces critical errors if any contaminants, such as dirt, dust, or other interfering substances or other anomalies, such as scratches, are in the reaction site. The errors arise because this approach cannot determine the features in an object, such as shape or size of particulates in the reaction site. Therefore, there is a need for an instrument with improved discriminations of features within the field of view of the imaging device. Although higher magnification and selective mask techniques may be developed for the photomultiplier tube to yield the desired selectivity, the cost, reliability and throughput of such a device would make it impractical. In addition, such devices do not produce an image to which a human technician is accustomed and therefore it would be difficult for a technician to score the results on that image to confirm the instrument generated results.
Therefore, in view of the above it is a primary object of the present invention to provide an apparatus and method for automatic processing of a qualitative, quantitative or morphological analysis of test specimens including serum, plasma or cellular components as well as other non-biological specimens.
It is another object of the present invention to provide an automated instrument for performing HLA typing, including automated cell preparation, automated sample processing, and automated reading of the results.
It is a further object of the present invention to provide an apparatus and method for performing an assay on a disposable or reusable cartridge on which the specimen to be analyzed may be placed and which will be analyzed by an automated instrument.
It is another object of the present invention to provide an analytical instrument with a liquid dispensing and liquid level detection system which can control liquid dispensing of very small volumes, accurately determine liquid levels even in liquids with a relatively low dielectric constant, and determine droplet formation and separation.
It is another object of the present invention to provide a detection system which can detect the interface between liquids with different dielectric constants.
It is another object of the present invention to provide an analyzing instrument with powerful, cost-effective and efficient image processing for automated sizing and counting of data.
It is yet another object of the present invention to provide an apparatus which is field upgradeable to perform different types of assays.